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New approaches for micropropagation and cryopreservation of Agave peacockii, an endangered species

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Abstract

More than 50% out of 129 of Agave species are endemic to Mexico. Among them, Agave peacockii is among the list of threatened species that require special protection. In this work, we aimed at developing new supplementary strategies to achieve micropropagation and perform cryopreservation of in vitro grown shoot-tips of A. peacockii. For multiplication, the addition of two cytokinins, 6-benzylaminopurine (26.6 μM) and kinetin (27.84 μM) to MS semisolid medium significantly favoured the morphogenetic response and produced the highest (87.00 ± 17.18) shoot generation number after 60 days of culture. This interaction was more effective than using the same growth regulators separately. Propagated and rooted plantlets were acclimatized with 100% survival and normal morphological development. For cryopreservation, an optimized protocol following droplet-vitrification allowed obtaining 98% and 96% regrowth before and after cryopreservation, respectively. Shoot-tips (1 mm in length × 1 mm wide) were excised of in vitro-propagated plants, subjected to preculture on MS semisolid medium with 0.3 M sucrose for 1d, loaded in solution with 0.4 M sucrose and 1.6 M glycerol for 20 min, exposed to Plant Vitrification Solution 2 for 15 min, and then, immersed in liquid nitrogen in droplets of PVS2 placed on aluminium foil strips. The vegetative growth of cryo-derived plants and of the in vitro propagated plants was compared under greenhouse conditions. No significant differences were detected in most assessed characteristics after 120 days of culture. The results presented here constitute new viable biotechnological approaches for the in vitro propagation and long-term conservation of endangered Agave germplasm.

Key message

Agave peacockii shoot micropropagation was induced combining 6-benzylaminopurine (BAP) and 6-furfuryl-aminopurine (kinetin). A droplet-vitrification protocol was optimized to cryopreserve shoot-tips. Greenhouse performance of in vitro and cryo-derived plants was similar.

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Abbreviations

KIN:

6-Furfuryl-aminopurine (kinetin)

BAP:

6-Benzylaminopurine

ANOVA:

Analysis of variance

PEG:

Polyethylene glycol

LN:

Liquid nitrogen

LSD:

Least significant difference

L2:

Vitamins Phillips and Collins (1979)

MS:

Medium Murashige and Skoog (1962)

PGRs:

Plant growth regulators

PVS:

Plant vitrification solution

2,4-D:

2,4-Dichlorophenoxyacetic acid

IBA:

Indole-3-butyric acid

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Acknowledgements

This work was supported by The Rufford Foundation (Grant 31255-2) and Consejo Nacional de Ciencia y Tecnología (Grant scholarship No. 291236).

Funding

This study has been supported by The Rufford Foundation (Grant 31255-2) and Consejo Nacional de Ciencia y Tecnología (Grant scholarship No. 291236).

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Authors and Affiliations

  1. Doctorado en Ciencias en Biosistemática, Ecología y Manejo de Recursos Naturales y Agrícolas, Centro Universitario de Ciencias Biológicas y Agropecuarias, Universidad de Guadalajara, Zapopan, Jalisco, Mexico

    Lourdes Delgado-Aceves & Felipe de Jesús Romo-Paz

  2. Departamento de Botánica y Zoología, Centro Universitario de Ciencias Biológicas y Agropecuarias, Universidad de Guadalajara, Zapopan, Jalisco, Mexico

    Liberato Portillo

  3. Huntington Library, Art Museum, and Botanical Gardens, San Marino, CA, USA

    Raquel Folgado

  4. Laboratorio de Biotecnología y Criobiología Vegetal, Facultad de Ciencias Químicas, Universidad Veracruzana, Orizaba, Veracruz, Mexico

    María Teresa González-Arnao

Authors
  1. Lourdes Delgado-Aceves
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  2. Liberato Portillo
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  3. Raquel Folgado
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  4. Felipe de Jesús Romo-Paz
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  5. María Teresa González-Arnao
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Contributions

L-DA and MT-GA wrote and reviewed the manuscript. L-DA, LP and FJ-RP designed and performed the shoot generation experiments. L-DA and LP conducted histological cuts, rooting and acclimatization experiments. L-DA, FJ-RP and RF developed the cryopreservation experiments. LD and MT-GA conducted the statistical analysis. All the authors read and approved the manuscript.

Corresponding author

Correspondence to María Teresa González-Arnao.

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Communicated by Ranjith Pathirana.

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Delgado-Aceves, L., Portillo, L., Folgado, R. et al. New approaches for micropropagation and cryopreservation of Agave peacockii, an endangered species. Plant Cell Tiss Organ Cult 150, 85–95 (2022). https://doi.org/10.1007/s11240-022-02246-z

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